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DMS footprinting of structured RNAs and RNA–protein complexes

Nature Protocols volume 2, pages 2608–2623 (2007)Cite this article

Abstract

We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA–protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nt). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as 'control' conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a 'footprint'), it is possible to detect local changes in the secondary and tertiary structure of RNA, as well as the formation of RNA–protein contacts. This protocol takes 1.5–3 d to complete, depending on the type of analysis used.

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Figure 1: General schematic diagram of DMS footprinting.
Figure 2: Establishment of a solution quench of DMS modification reaction.
Figure 3: DMS footprinting of an RNP and quantitative analysis using manual boxing.
Figure 4: Quantitative analysis of DMS footprinting to monitor RNA folding.
Figure 5: DMS footprinting gel from an experiment in which the RNA degradation steps were omitted (Steps 25 and 26).

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Acknowledgements

We thank Yaqi Wan for assistance in preparing Figure 4 and Liz Doherty and Jennifer Doudna for early advice on footprinting and for sharing their protocol. Research in our labs is supported by grants from the NIH (R01-GM070456 to R.R. and R01-GM037951 to Alan M. Lambowitz) and the Welch Foundation (F-1563 to R.R.).

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  1. Department of Chemistry and Biochemistry and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, 78712, Texas, USA

    Pilar Tijerina, Sabine Mohr & Rick Russell

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  1. Pilar Tijerina
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Correspondence to Rick Russell.

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Tijerina, P., Mohr, S. & Russell, R. DMS footprinting of structured RNAs and RNA–protein complexes. Nat Protoc 2, 2608–2623 (2007). https://doi.org/10.1038/nprot.2007.380

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